U.S. patent application number 13/496313 was filed with the patent office on 2012-07-19 for inhibition of endosomal toll-like receptor activation.
Invention is credited to Bruce A. SULLENGER.
Application Number | 20120183564 13/496313 |
Document ID | / |
Family ID | 43759220 |
Filed Date | 2012-07-19 |
United States Patent
Application |
20120183564 |
Kind Code |
A1 |
SULLENGER; Bruce A. |
July 19, 2012 |
INHIBITION OF ENDOSOMAL TOLL-LIKE RECEPTOR ACTIVATION
Abstract
The present invention relates, in general, to
pattern-recognition receptors (PRRs), including toll-like receptors
(TLRs), and, in particular, to a method of inhibiting nucleic
acid-induced activation of, for example, endosomal TLRs using an
agent that binds to the nucleic acid ("nucleic acid binding
agent"), preferably, in a manner that is independent of the
nucleotide sequence, the chemistry (e.g., DNA or RNA, with or
without base or sugar modifications) and/or the structure (e.g.,
double-stranded or single-stranded, complexed or uncomplexed with,
for example protein) of the nucleic acid(s) responsible for
inducing TLR activation. The invention also relates to methods of
identifying nucleic acid binding agents suitable for use in such
methods.
Inventors: |
SULLENGER; Bruce A.;
(US) |
Family ID: |
43759220 |
Appl. No.: |
13/496313 |
Filed: |
September 16, 2010 |
PCT Filed: |
September 16, 2010 |
PCT NO: |
PCT/US10/02516 |
371 Date: |
March 15, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61243090 |
Sep 16, 2009 |
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Current U.S.
Class: |
424/172.1 ;
435/6.1; 514/1.1; 514/20.9; 514/410; 514/55; 514/616; 514/674 |
Current CPC
Class: |
A61P 11/00 20180101;
A61K 38/1709 20130101; A61K 38/16 20130101; A61P 25/00 20180101;
A61K 31/70 20130101; A61P 9/00 20180101; A61P 7/02 20180101; A61P
31/00 20180101; A61K 31/785 20130101; A61P 29/00 20180101; A61P
35/00 20180101; A61P 17/06 20180101; A61K 31/13 20130101; A61P
19/02 20180101; A61P 31/04 20180101; A61P 3/04 20180101; A61K
31/722 20130101 |
Class at
Publication: |
424/172.1 ;
514/1.1; 514/20.9; 514/674; 514/55; 514/616; 514/410; 435/6.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 38/14 20060101 A61K038/14; A61K 31/785 20060101
A61K031/785; A61K 31/722 20060101 A61K031/722; A61K 31/16 20060101
A61K031/16; A61K 31/407 20060101 A61K031/407; C12Q 1/68 20060101
C12Q001/68; A61P 29/00 20060101 A61P029/00; A61P 35/00 20060101
A61P035/00; A61P 31/00 20060101 A61P031/00; A61P 9/00 20060101
A61P009/00; A61P 31/04 20060101 A61P031/04; A61P 25/00 20060101
A61P025/00; A61P 19/02 20060101 A61P019/02; A61P 11/00 20060101
A61P011/00; A61P 3/04 20060101 A61P003/04; A61P 17/06 20060101
A61P017/06; A61P 7/02 20060101 A61P007/02; A61K 38/02 20060101
A61K038/02 |
Goverment Interests
[0002] This invention was made with government support under Grant
No. HL65222 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of inhibiting nucleic acid-induced activation of a
pattern recognition receptor (PRR) comprising administering to a
patient in need thereof an agent that binds a nucleic acid
responsible for said induction of activation in an amount and under
conditions such that inhibition of said activation is effected.
2. The method according to claim 1 wherein said PRR is an endosomal
toll-like receptor (TLR) or a cytoplasmic PRR.
3. The method according to claim 1 wherein said agent binds said
nucleic acid in a manner that is independent of nucleotide
sequence, chemistry or structure.
4. The method according to claim 1 wherein said agent is a
positively charged compound.
5. The method according to claim 4 wherein said compound is a
protein, polypeptide, peptide, lipid or natural or synthetic
polymer.
6. The method according to claim 5 wherein said compound is a
protein, polypeptide or peptide.
7. The method according to claim 6 wherein said compound is a
protamine, a DNA or RNA reactive antibody, a heterogeneous nuclear
ribonucleoprotein, a chimeric peptide, or a viral protein that
packages DNA or RNA.
8. The method according to claim 5 wherein said compound is a
cationic lipid.
9. The method according to claim 8 wherein said cationic lipid is
linear poly(ethyleneimine) (PEI), poly(L-lysine) (PLL),
poly(amidoamine) (PAMAM) dendrimer generation 4, chitosan, DOTMA,
DOTAP, DMRIE, DOTIM, DOGS, DC-Chol, BGTC or DOPE.
10. The method according to claim 1 wherein said agent is an
intercalating agent or a porphyrin.
11. The method according to claim 1 wherein said agent is
polycationic polymer.
12. The method according to claim 11 wherein said polycationic
polymer is CDP, CDP-im, PPA-DPA, PAMAM or HDMBr.
13. A method of controlling an autoimmune or inflammatory response
comprising administering to a patient in need thereof an agent that
binds a nucleic acid responsible for said response in an amount and
under conditions such that said response is controlled.
14. The method according to claim 13 wherein said nucleic acid is
pathogen derived or released from a dead or damaged cell of said
patient.
15. The method according to claim 14 wherein said patient suffers
from an infectious disease, a cardiovascular disease, cancer,
bacterial sepsis, multiple sclerosis, systemic lupus erythematosis,
rheumatoid arthritis, COPD, obesity or psoriasis.
16. A method of preventing the induction of, or inhibiting the
progression of, a thrombotic disorder comprising administering to a
patient in need thereof an agent that binds a nucleic acid
responsible for said induction or progression in an amount and
under conditions such that said prevention or inhibition is
effected.
17. A method of identifying a candidate nucleic acid binding agent
suitable for use in the method according to claim 1 comprising: i)
culturing PRR-containing cells with a first PRR agonist in the
presence and absence of a test agent, ii) obtaining a supernatant
sample from said culture of step (i), iii) analyzing said sample
for the presence of a product of an intracellular signaling event
initiated by PRR activation, and iv) repeating steps (i)-(iii) with
second PRR agonist having a sequence, chemistry or structure
different from that of the first agonist, wherein a test agent that
inhibits PRR agonist activation in a manner independent of
sequence, chemistry or structure of the PRR agonist used is a
candidate nucleic acid binding agent.
Description
[0001] This application claims priority from U.S. Provisional
Application No. 61/243,090, filed Sep. 16, 2009, the entire content
of which is incorporated herein by reference.
TECHNICAL FIELD
[0003] The present invention relates, in general, to
pattern-recognition receptors (PRRs), including toll-like receptors
(TLRs), and, in particular, to a method of inhibiting nucleic
acid-induced activation of, for example, endosomal TLRs using an
agent that binds to the nucleic acid ("nucleic acid binding
agent"), preferably, in a manner that is independent of the
nucleotide sequence, the chemistry (e.g., DNA or RNA, with or
without base or sugar modifications) and/or the structure (e.g.,
double-stranded or single-stranded, complexed or uncomplexed with,
for example protein) of the nucleic acid(s) responsible for
inducing TLR activation. The invention also relates to methods of
identifying nucleic acid binding agents suitable for use in such
methods.
BACKGROUND
[0004] TLRs are type I transmembrane proteins composed of an
extracellular domain of leucine-rich repeats and an intracellular
Toll/interleukin-1 (IL-1) receptor (TIR) domain (Leulier and
Lemaitre, Nat. Rev. Genet. 9:165-178 (2008)). Ten human and twelve
mouse TLRs have been identified. Each TLR is able to recognize a
particular molecular pattern. For instance, TLR2, 4, 5, 6 and 11
bind to bacterial outer membrane molecules such as
lipopolysaccharide (LPS), peptidoglycan and lipoteic acid while
TLR3, TLR7, TLR8 and TLR9 recognize bacterial, viral or even
endogenous nucleic acids (Kawai and Akira, Semin. Immunol. 19:24-32
(2007)). Moreover, TLRs can be classified based on their cellular
localization: TLR1, 2, 4, 5 and 6 are expressed on the cell surface
while TLR3, 7, 8 and 9 are localized mostly, though not
exclusively, in endosomal compartments (Kawai and Akira, Semin.
Immunol. 19:24-32 (2007)).
[0005] When pathogens invade a host, innate immune cells such as
macrophages, neutrophils, natural killer cells and dendritic cells
recognize pathogen-associated molecular patterns (PAMPs) and
endogenous damage-associated molecular patterns (DAMPs) through
TLRs. TLR activation initiates intracellular signaling events that
result in the expression of immune response genes including
inflammatory and immune modulatory cytokines, chemokines, immune
stimulatory receptors, which augments killing of pathogens and
initiates the process of developing acquired immunity (Takeda and
Akira, Int. Immunol. 17:1-14 (2005), Akira et al, Cell 124:783-801
(2006)). Inappropriate activation of some members of the TLR
family, on the other hand, contribute to development of a variety
of diseases including bacterial sepsis (TLR1, TLR2, TLR3, TLR4 and
TLR9) (Wurfel et al, Am. J. Respir. Crit. Care Med. 178:710-720
(2008), Knuefermann et al, Circulation 110:3693-3698 (2004),
Cavassani et al, J. Exp. Med. 205:2609-2621 (2008), Alves-Filho et
al, Crit. Care Med. 34:461-470 (2006), Tsujimoto et al, J. Hepatol.
45:836-843 (2006)), non-infection systemic inflammatory response
syndrome (TLR4) (Breslin et al, Shock 29:349-355 (2008)), multiple
sclerosis (TLR3, TLR4 and TLR9) (Chen et al, Int. Immunopharmacol
7:1271-1285 (2007)), systemic lupus erythematosus (SLE) (TLR7 and
TLR9) (Marshak-Rothstein and Rifkin, Annu. Rev. Immunol. 25:419-441
(2007)) and rheumatoid arthritis (TLR3, TLR4, TLR7, TLR8 and TLR9)
(Choe et al, J. Exp. Med. 197:537-542 (2003), O'Neil, Nat. Clin.
Pract. Rheumatol. 4:319-327 (2008)). Moreover, preclinical and
clinical studies indicate that inhibition of TLR activity has
therapeutic benefits for treating certain diseases. For example,
diverse LPS-neutralizing agents and TLR4 antagonists have been
evaluated to treat inflammatory diseases in animal and clinical
studies (Leon et al, Pharm. Res. 25:1751-1761 (2008)). A TLR9
inhibitor, inhibitory CpG DNA (Plitas et al, J. Exp. Med.
205:1277-1283 (2008)), and an antagonistic anti-TLR3 antibody
(Cavassani et al, J. Exp. Med. 205:2609-2621 (2008)) enhanced
survival of a mouse with polymicrobial sepsis.
Oligonucleotide-based TLR7 and TLR9 inhibitors prevented IFN.alpha.
production from human plasmacytoid dendritic cells stimulated with
serum from SLE patients (Barrat et al, J. Exp. Med. 202:1131-1139
(2005)). Unfortunately, the redundancy of the TLR family may limit
the utility of inhibitors that target individual TLRs.
[0006] Upon stimulation, all TLRs recruit intracellular
TIR-domain-containing adapters, such as TRIF and MyD88 (Kawai and
Akira, Semin. Immunol. 19:24-32 (2007)). These adapter molecules
mediate a downstream cascade of TLR-associated signaling. TRIF is
recruited to TLR3 and TLR4, and appears to activate IRF3, MAPK, and
NF-.kappa.B while MyD88 is associated with all TLRs, except TLR3,
and phosphorylates IRAK, IRF5, IRF7, MAPK and NF-.kappa.B, which
enhance the expression of type I IFN, inflammatory cytokine and
IFN-inducible genes (Kawai and Akira, Semin. Immunol. 19:24-32
(2007)). Unlike other TLRs, endosomal TLRs, TLR3, 7, 8 and 9, all
recognize microbial or host nucleic acids, as PAMPs or DAMPs,
respectively. The redundancy and interconnectedness of the TLR
signaling pathway suggests that it will be important to inhibit the
activity of multiple TLRs simultaneously to effectively control
inflammatory and autoimmune responses and to enhance the clinical
efficacy of TLR antagonists as therapeutic agents.
[0007] It was discovered recently that certain cationic polymers
are able to counteract the activity of a variety of
oligonucleotide-based drugs (e.g., aptamers), irrespective of their
nucleotide sequences (Oney et al, Control of Aptamer Activity by
Universal Antidotes: An Approach to Safer Therapeutics, Nature
Medicine (in press)). Moreover, immune stimulatory siRNA, a TLR7
agonist, condensed with a cyclodextrin-based polymer has been shown
not to activate TLR7 (Hu-Lieskovan et al, Cancer Res. 65:8984-8992
(2005)). The present invention results, at least in part, from
studies designed to determine whether agents that bind DNAs and
RNAs in a sequence-independent manner (e.g., nucleic acid-binding
cationic polymers) can neutralize endosomal TLR ligands and thereby
inhibit activation of the corresponding TLRs.
SUMMARY OF THE INVENTION
[0008] The present invention relates generally to PRRs, including
TLRs (e.g., endosomal TLRs). More specifically, the invention
relates to a method of inhibiting nucleic acid-induced activation
of, for example, endosomal TLRs using an agent that binds to the
nucleic acid ("nucleic acid binding agent"), preferably, in a
manner that is independent of the nucleotide sequence, the
chemistry (e.g., DNA or RNA, with or without base or sugar
modifications) and/or the structure (e.g., double-stranded or
single-stranded, complexed or uncomplexed with, for example
protein) of the nucleic acid responsible for inducing TLR
activation. The invention further relates to methods of controlling
inflammatory and/or autoimmune responses resulting from nucleic
acid-induced receptor (e.g. endosomal TLR) activation using such a
nucleic acid binding agent. The invention further relates to
methods of identifying nucleic acid binding agents suitable for use
in such methods.
[0009] Objects and advantages of the present invention will be
clear from the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIGS. 1A and 1B. Cationic polymers inhibit nucleic acid
induced activation of TLR3 and TLR9. (FIG. 1A) The murine
macrophage cell line, Raw264.7 was co-incubated in a 24-well
microplate with a TLR9 agonist (CpG) (2 .mu.M), a TLR3 agonist
(poly I:C) (10 .mu.g/ml) or a TLR4 agonist (LPS) (100 ng/ml) along
with the cationic polymers, CDP, HDMBr, PAMAM, poly L-lysine or
protamine (20 .mu.g/ml) or PBS. Unmethylated GpC ODNs were used as
a negative control for CpG. After 18-hours of incubation, culture
supernatants were collected and analyzed for cytokines by ELISA.
(FIG. 1B) The treated cells were tested for their expression of the
co-stimulatory molecule CD86 using FACS. The light blue line
represents PBS-treated cells. Green and red lines represent GpC-
and CpG-treated cells, respectively. Data represents three
individual experiments. Error bar is S.D.; n=3. *P<0.005 (both
TNF.alpha. and IL-6; CpG or poly I:C+Cationic polymers vs CpG or
Poly I:C alone); .dagger-dbl. P=0.0169 and 0.0395 (TNF.alpha. and
IL-6, respectively; poly I:C+CDP vs poly I:C alone); P=0.0256 and
0.0281 (TNF.alpha. and IL-6, respectively; poly I:C+protamine vs
poly I:C alone).
[0011] FIGS. 2A and 2B. Timing of cationic polymer mediated
inhibition of TLR activation. (FIG. 2A) Cells were incubated with
CpG (2 .mu.M) in a 24-well microplate. CDP (20 .mu.g/ml) was added
at 0, 1/2, 1, 2, 4, 8 or 12 hours following the addition of CpG. At
24 hours after CpG treatment culture supernatants were collected
and analyzed for TNF.alpha. and IL-6 production. (FIG. 2B) Cells
were pre-incubated for 1 or 2 hours with CDP or PBS, washed three
times with complete medium and then incubated in culture media
supplemented with CpG. Simultaneous treatment of cells with CpG and
CDP was used as a control. At 5 hours after CpG treatment the
amount of TNF.alpha. in the culture supernatants were measured by
ELISA. Error bar is S.D.; n=3. *P<0.0001 (both TNF.alpha. and
IL-6; CpG+CDP vs CpG alone); .dagger-dbl. P=0.0230 and <0.0001
(TNF.alpha. and IL-6, respectively; CpG+CDP vs CpG alone); P=0.0257
and 0.0003 (TNF.alpha. and IL-6, respectively; CpG+CDP vs CpG
alone).
[0012] FIGS. 3A and 3B. Dose-dependent inhibition of cationic
molecules on TLR3 and TLR9 activation. 1.times.10.sup.6 Raw264.7
cells were cultured for 18 hours with either CpG (1 .mu.M) (FIG.
3A) or poly I:C (10 .mu.g/ml) (FIG. 3B) in the presence or absence
of CDP (.quadrature.), HDMBr () or PAMAM (.box-solid.) at the
indicated concentration. Amounts of TNF.alpha. and IL-6 in the
culture supernatant were measured by ELISA. Error bar is S.D.; n=3.
NT: not tested.
[0013] FIGS. 4A-4C. TLR3- or TLR9-mediated acute liver inflammation
can be alleviated by nucleic acid-binding polymers. (FIG. 4A) Mice
(5-10 mice/group) were i.p. injected with D-GalN (20 mg) alone, CpG
(51 .mu.g) alone, D-GalN+GpC (51 .mu.g) or D-GalN+CpG (51 .mu.g).
After 5-10 minutes, PBS (100 .mu.l), CDP (200 .mu.g; blue diamond),
HDMBr (200 or 400 .mu.g; red triangle) or PAMAM (200 or 400 .mu.g;
green rectangle) was administered i.p. into mice challenged with
D-GalN+CpG. Mice were monitored daily for survival. (FIG. 4B)
Mixture of Poly I:C (200 .mu.g) and D-GalN (20 mg) in PBS (100
.mu.l) was injected i.p. into mouse (5 mice/group). Subsequently,
PBS (100.mu.l; black circle), CDP (400 or 800 .mu.g; blue diamond),
HDMBr (200 or 400 .mu.g; red triangle) or PAMAM (200 or 400 .mu.g;
green rectangle) was injected i.p. There is 5-10 minutes interval
between injections. (FIG. 4C) Mice were injected with PBS,
CpG+D-GalN or CpG+D-GalN+CDP. Sixteen hours following injection,
liver specimens were collected for histological studies
(hematoxylin and eosin staining). A representative of three
individual results. Magnification .times.20.
[0014] FIGS. 5A and 5B. Stoichiometry of TLR inhibition of CDP.
Raw264.7 cells were cultured for 18 hours with either CpG (1 .mu.M,
2 .mu.M, 4 .mu.M, 8 .mu.M) (FIG. 5A) or poly I:C (10 .mu.g/ml or 25
.mu.g/ml) (FIG. 5B). TLR ligands were simultaneously supplemented
with CDP at various concentration (0, 4, 8, 12, 16, 20, 24, 36, 48
.mu.g/ml for CpG; 0, 10, 20, 30,40, 80, 160 .mu.g/ml for poly I:C).
Amount of TNF.alpha. was measured by ELISA. % inhibition was
calculated by ([CpG or poly I:C]-[CpG or poly I:C+CDP])/[CpG or
poly I:C].times.100.
[0015] FIG. 6. Cellular toxicity of cationic molecules.
1.times.10.sup.6 Raw264.7 cells were cultured for 24 hours with CDP
(black), HDMBr (red), PAMAM (blue), PPA-DPA (green), protamine
(gray) or poly L-lysine (purple) at various concentration (10, 20,
40, 80, 160, 280, 400 and 600 .mu.g/ml). Viability of cells was
analyzed using hematocytometer after staining with trypan blue
(Sigma, St. Louis, Mo.).
[0016] FIGS. 7A-7D. CDP enhanced CpG uptake of cells. Raw264.7
cells (1.times.10.sup.5 cells/well) were cultured overnight in
8-well chamber slide (Nalge Nunc International Corp, Naperville,
Ill.). After thrice washing with cold complete media, cells were
replenished with fresh complete media including 1 .mu.M of CpG
conjugated with 6-FAM at 5' end with or without 10 .mu.g/ml of CDP.
Cells were incubated for 1 (FIGS. 7A and 7C) or 2 hours (FIGS. 7B
and 7D) at either 4.degree. C. or 37.degree. C. Fluoresce signals
were observed with the Olympus IX71 Inverted Microscope (Olympus,
Center Valley, Pa.). The images were analyzed using the Olympus DP
Controller Ver.1.2.1.108. Data represents two individual
experiments. Magnification is 40.times..
DETAILED DESCRIPTION OF THE INVENTION
[0017] PRRs are a pivotal component of host immune cells to protect
tissues from various harmful stimuli, such as pathogens and damaged
cells. A variety of PRRs, including RIG-I-like receptors (RLRs),
dsRNA-dependent protein kinase R (PKR), DNA-dependent activator of
IRFs (DAI) and TLRs can recognize diverse products of pathogens and
damaged cells that are referred to PAMPs and DAMPs (Lotze et al,
Immunol. Reviews 220:60-81 (2007)).
[0018] TLRs play a central role in host innate and acquired
immunity, as well as in the pathogenesis of various diseases,
including infectious diseases, inflammatory diseases and autoimmune
diseases. TLRs 3, 7, 8 and 9 are localized in endosomes can be
activated by microbial and host nucleic acids.
[0019] The present invention relates, in one embodiment, to a
method of inhibiting nucleic acid-induced activation of endosomal
TLRs. The method comprises administering to a patient in need
thereof an agent that binds nucleic acids responsible for induction
of TLR activation in an amount and under conditions such that
inhibition of that activation is effected. Advantageously, the
agent binds the nucleic acids in a manner that is independent of
the nucleotide sequence, the chemistry (e.g., DNA or RNA, with or
without base or sugar modifications) and/or the structure (e.g.,
double-stranded or single-stranded, complexed or uncomplexed with,
for example, a protein) of the nucleic acids responsible for
inducing TLR activation. The present method can be used to treat
inflammatory and/or autoimmune responses resulting from endosomal
activation.
[0020] Nucleic acid binding (scavenging) agents of the invention
include pharmaceutically acceptable member(s) of a group of
positively charged compounds, including proteins, lipids, and
natural and synthetic polymers, that can bind nucleic acids in, for
example, biologically fluids.
[0021] Proteinaceous nucleic acid binding agents of the invention
include protamines, a group of proteins that yield basic amino
acids on hydrolysis and that occur combined with nucleic acid in
the sperm of fish, such as salmon. Protamines are soluble in water,
are not coagulated by heat, and comprise arginine, alanine and
serine (most also contain proline and valine and many contain
glycine and isoleucine). In purified form, protamine has been used
for decades to neutralize the anticoagulant effects of heparin.
Nucleic acid binding agents of the invention also include protamine
variants (e.g., the +18RGD variant (Wakefield et al, J. Surg. Res.
63:280 (1996)) and modified forms of protamine, including those
described in Published U.S. Application No. 20040121443. Other
nucleic acid binding agents of the invention include protamine
fragments, such as those described in U.S. Pat. No. 6,624,141 and
U.S. Published Application No. 20050101532. Nucleic acid binding
agents of the invention also include, generally, peptides that
modulate the activity of heparin, other glycosaminoglycans or
proteoglycans (see, for example, U.S. Pat. No. 5,919,761). The
invention further includes pharmaceutically acceptable salts of the
above-described nucleic acid binding agents, as appropriate,
including sulfate salts.
[0022] Proteinaceous nucleic acid binding agents of the invention
also include DNA and/or RNA reactive antibodies. For example,
anti-nuclear antibodies, such as those indicative of lupus
erythematosis, Sjogren's syndrome, rheumatoid arthritis, autoimmune
hepatitis, scleroderma, polymyositis and dermatomyositis, can be
used. Specific examples of antibodies that recognize RNA/DNA
include those described by Kitagawa et al (Mol. Immunol.
19(3):413-20 (1982)), Boguslawski et al (J. Immunol. Methods
89(1):123-30 (1986)), Williamson et al (Proc. Natl. Acad. Sci.
98(4):1793-98 (2001)), and Blanco et al (Clin. Exp. Immunol.
86(1):66-70 (1991)).
[0023] In addition, heterogeneous nuclear ribonucleoproteins
(HNRPs) can also be used in accordance with the invention.
Cationic, peptides that bind nucleic acids (e.g., in a
sequence-independent manner) are suitable for use. For example, a
chimeric peptide synthesized by adding nonamer arginine residues at
the carboxy terminus of RVG (to yield RVG-9R) has been described by
Kumar et al (Nature 448:39-43 (2007)). Viral proteins that package
(e.g., coat) DNA or RNA (e.g., HIV gag protein), and peptides
derived therefrom, can also be used in the present methods.
[0024] Cationic lipids can also be used as nucleic acid binding
agents in accordance with the invention. Suitable cationic lipids
include those described by Morille et al (Biomaterials 29:3477
(2008)) (e.g., linear poly(ethyleneimine) (PEI), poly(L-lysine)
(PLL), poly(amidoamine) (PAMAM) dendrimer generation 4, chitosan,
DOTMA, DOTAP, DMRIE, DOTIM, DOGS, DC-Choi, BGTC and DOPE).
[0025] Nucleic acid binding agents of the invention also include
intercalating agents. Examples include ethidium bromide,
proflavine, daunomycin, doxorubicin and thalidomide. Nucleic acid
binding porphyrins can also be used in accordance with the
invention (see Table 1).
[0026] Preferred nucleic acid binding agents of the invention
include polycationic polymers. Preferred polycationic polymers
include biocompatible polymers (that is, polymers that do not cause
significant undesired physiological reactions) that can be either
biodegradable or non-biodegradable polymers or blends or copolymers
thereof Examples of such polymers include, but are not limited to,
polycationic biodegradable polyphosphoramidates, polyamines having
amine groups on either the polymer backbone or the polymer side
chains, nonpeptide polyamines such as poly(aminostyrene),
poly(aminoacrylate), poly(N-methyl aminoacrylate),
poly(N-ethylaminoacrylate), poly(N,N-dimethyl aminoacrylate),
poly(N,N-diethylaminoacrylate), poly(aminomethacrylate),
poly(N-methyl amino-methacrylate), poly(N-ethyl aminomethacrylate),
poly(N,N-dimethyl aminomethacrylate), poly(N,N-diethyl
aminomethacrylate), poly(ethyleneimine), polymers of quaternary
amines, such as poly(N,N,N-trimethylaminoacrylate chloride),
poly(methyacrylamidopropyltrimethyl ammonium chloride); natural or
synthetic polysaccharides such as chitosan, cyclodextrin-containing
polymers, degradable polycations such as
poly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA); polycationic
polyurethanes, polyethers, polyesters, polyamides, polybrene, etc.
Particularly preferred cationic polymers include CDP, CDP-im,
PPA-DPA, PAMAM and HDMBr.
[0027] Nucleic acid binding agents of the invention can include
compounds of types described in Table 1, or derivatives thereof.
Several of the compounds described in Table 1 contain cationic-NH
groups permitting stabilizing charge-charge interactions with a
phosphodiester backbone. Nucleic acid binding agents of the
invention containing secondary amines can include, for example,
5-350 such groups (e.g., 5-300, 5-250, 5-200, 5-100, 5-50, 50-100,
50-200, 50-300, 50-350, 100-200, 100-300, 100-350, 200-350,
200-300, or 250-350), and can have a molecular weight in the range
of, for example, 2,000 to 50,000 (e.g., 10,000 to 50,000 or 20,000
to 40,000).
TABLE-US-00001 TABLE 1 Compound Abbreviation Molecular structure
Remark Poly-L-lysine PLL ##STR00001## 1. Commercially available. 2.
Carbonyl moiety (--C.dbd.O) which could permit additional
stabilization to the complex through hydrogen bonds with DNA.
Poly-L-ornithine PLO ##STR00002## 1. Commercially available. 2.
Carbonyl moiety (--C.dbd.O) which could permit additional
stabilization to the complex through hydrogen bonds with DNA.
Polyphosphoramidate polymer series PPA-SP PPA-BA PPA-EA PPA-MEA
PPA-DMA PPA-DEA PPA-TMA PPA-DPA ##STR00003## PPA 1. Polymers with
an identical backbone but different side chains ranging from
primary to quaternary amines. Provide a platform for a systematic
study. ##STR00004## PPA-SP 2. Lower cytotoxicity compared with
polyethylenimine (PEI) and poly-L- lysine (PLL).
--NH--(CH.sub.2).sub.4--NH.sub.2 PPA-BA
--NH--(CH.sub.2).sub.2--NH.sub.2 PPA-EA
--NH--(CH.sub.2).sub.2--NH--CH.sub.3 PPA-MEA ##STR00005## PPA-DMA
##STR00006## PPA-DEA ##STR00007## PPA-TMA ##STR00008## PPA-DPA
Polyphosphoramidate diprophylamine- poly ethylene glycol copolymer
PPA-DPA-b- PEG.sub.2000 ##STR00009## 1. a copolymer of PPA-DPA and
PEG. Polyethyleneimine PEI ##STR00010## 1. Commercially available.
2. PEI with branched structure condenses DNA to a greater extent
than linear ones. 3. high cytotoxicity. Ionene e.g. polybrene
##STR00011## 1. Commercially available. 2. Have high charge
density. Natural polyamine H.sub.2N--(CH.sub.2).sub.4--NH.sub.2 1.
Commercially e.g.
H.sub.2N--(CH.sub.2).sub.3--NH--(CH.sub.2).sub.4--NH--(CH.sub.2).sub-
.3--NH.sub.2 available. Putrescine
H.sub.2N--(CH.sub.2).sub.4--NH--(CH.sub.2).sub.3--NH.sub.2 2. The
most extensive Spermine work Spermidine on their binding with DNA
has been carried out and have remarkable effects on the DNA
condensation. Poly(allylamine) PAL ##STR00012## 1. Commercially
available. 2. Highly positive charged 3. Low toxicity. Peptide
nucleic acid PNA ##STR00013## 1. Commercially available. 2. Binding
through Watson-crick base pairing, thus binding is typically
stronger and more rapid. Water soluble porphyrin e.g. poly tetra(p-
aminophenyl) porphyrin poly tetra (methylpyridine) porphyrin
H.sub.2TAPP H.sub.2TMPyP.sub.4 ##STR00014## 1. Commerically
available. 2. One or two --N.sup.+(CH.sub.3).sub.3 groups of one
TAPP molecule bind with the phosphate groups. 3. The stacking of
TAPP along the surface of DNA leads to a favorite binding. 4.
Especially good binding with G- quadruplex through pi- pi
interaction. ##STR00015## Poly(porphyrin) or Porphyrin ladder e.g.
poly (H.sub.2 (p-TAPP) poly(por) A-AN)) ##STR00016## ##STR00017##
Poly (N,N- dimethylacrylamide) PDMA ##STR00018## Poly (2-
Methacryloyloxyethyl phosphorylcholine) PMPC ##STR00019##
Dendrimers e.g. polyamidoamine dendrimer PAMAM Dendrimer G2
##STR00020## 1. Commercially available. 2. Branched spherical shape
and a high density surface charge. 3. Low cytotoxicity. e.g.
polypropyl- eneimine dendrimer PPI dendrimer ##STR00021## 1. A
class of amine- terminated polymers, demonstrated to be efficient
gene delivery vectors. 2. Low cytotoxicity in a wide range of
mammalian cell lines. 3. Unique molecular structures, with defined
molecular weight, surface charge and surface functionality. These
properties of dendrimers provide a platform for a systematic study.
Partially deacetylated Chitin ##STR00022## 1. Commercially
available. Cyclodextrin grafted branched PEI or linear PEI
(.alpha.-CD: six sugar ring .beta.-CD: seven sugar ring .gamma.-CD:
eight sugar ring) CD-bPEI CD-lPEI ##STR00023## 1. Their IC.sub.50's
are 2-3 orders of magnitude higher than the corresponding non-
cyclodextrin-based polymer. ##STR00024## ##STR00025## Cyclodextrin
Containing Polymers CDP ##STR00026## CDP-Im ##STR00027##
[0028] Advantageously, the binding affinity of a nucleic acid
binding agent of the invention for a nucleic acid, expressed in
terms of Kd, is in the pM to .mu.M range, preferably, less than or
equal to 50 nM; expressed in terms of binding constant (K), the
binding affinity is advantageously equal to or greater than
10.sup.5M.sup.-1, preferably, 10.sup.5M.sup.-1 to 10.sup.8M.sup.-1,
more preferably, equal to or greater than 10.sup.6M.sup.-1. Thus,
the binding affinity of the sequence-independent nucleic acid
binding agents can be, for example, about 1.times.10.sup.5M.sup.-1,
5.times.10.sup.5 M.sup.-1, 1.times.10.sup.6M.sup.-1,
5.times.10.sup.6M.sup.-1, 1.times.10.sup.7 M.sup.-1,
5.times.10.sup.7 M.sup.-1; or about 10 pM, 100 pM, 1 nM, 10 nM, 100
nM, 1 .mu.M, 10 .mu.M, 100 .mu.M. "K" and "Kd" can be determined by
methods known in the art, including surface plasmon resonance or a
real time binding assay such as Biacore.
[0029] Preferred nucleic acid binding agents of the invention
simultaneously limit the activation of multiple endosomal TLRs
(e.g., TLR3 and TLR9). Particularly preferred are CDP or CDP-im,
HDMBr and PAMAM (see U.S. Pat. Nos. 7,270,808, 7,166,302,
7,091,192, 7,018,609, 6,884,789, 6,509,323, 5,608,015, 5,276,088,
5,855,900, U.S. Published Appln. Nos. 20060263435, 20050256071,
200550136430, 20040109888, 20040063654, 20030157030, Davis et al,
Current Med. Chem. 11(2) 179-197 (2004), and Comprehensive
Supramolecular Chemistry vol. 3, J. L. Atwood et al, eds, Pergamon
Press (1996)).
[0030] As indicated above, the present invention provides a method
of controlling (inhibiting or preventing) autoimmune and/or
inflammatory responses associated with activation of TLRs (e.g.,
endosomal TLRs such as TLR3 and TLR9). Such responses play a role
in the pathogensis of diseases/disorders that are associated with
presence in the circulation of the patient of free nucleic acids,
either pathogen-derived (e.g., viral- or bacterial-derived) nucleic
acids or nucleic acids released from dead or damaged host cells.
Specific diseases/disorders that can be treated using nucleic acid
binding agents of the invention include infectious diseases,
cardiovascular disease, cancer, bacterial sepsis, multiple
sclerosis, systemic lupus erythematosis, rheumatoid arthritis,
COPD, obesity and psoriasis.
[0031] RLRs are a family of cytoplasmic RNA helicases including
retinoic-acid-inducible protein I (RIG-I) and
melanoma-differentiation-associated gene 5 (MDA5). RIG-I recognize
uncapped 5'-triphosphate ssRNA and short dsRNA while MDA5 recognize
long dsRNA (Pichlmair et al, Science 314:997-1001 (2006), Hornung
et al, Science 314:994-997 (2006), Kato et al, J. Exp. Med.
205:1601-1610 (2008)). Signaling of RLRs is initiated by
interaction of caspase recruitment domain (CARD)-containing adapter
molecule, IFN.beta. promoter stimulator-1 (IPS-1), and induce
production of type I IFN and inflammatory cytokines (Kawai et al,
Ann. N.Y. Acad. Sc. 1143:1-20 (2008)). PKR is an IFN-inducible
cytosolic enzyme and recognizes viral dsRNAs while DAI recognizes
cytoplasmic dsDNA (Langland et al, Virus Res 119:100-110 (2006),
Takaoka et al, Nature 448:501-505 (2007)). These cytoplasmic PRRs,
including RIG-I, MDA5 and PKR, are able to recognize RNAs or DNAs
and activation of these PRRs is associated with type I IFN
production. Although their involvement in the pathogenesis of
inflammatory and autoimmune diseases has not been fully elucidated,
the cytoplasmic nucleic acid-sensing PRRs may also contribute to
the pathogenesis of such diseases because signaling from these
receptors robustly elicits production of IFNa, one of the major
pathogenic factors in a variety of inflammatory diseases (J.
Banchereau et al, Immunity 20:539-550 (2004)). Therefore, the
present invention also relates a method of inhibiting nucleic-acid
induced activation of these members of the PRR family using the
approaches and agents described above.
[0032] Another application of nucleic acid-binding agents
(scavengers) described herein is to counteract the effects of DNA
and RNA molecules that are released from cells and subsequently
induce thrombosis (Kannemeier et al, Proc. Natl. Acad. Sci.
104:6388-6393 (2007); Fuchs et al, Proc. Natl. Aad. Sci. Published
Online before Print Aug. 23, 2010). Recently it has been observed
that RNA and DNA molecules can activate the coagulation pathway as
well as platelets and thereby engender blood clotting (Kannemeier
et al, Proc. Natl. Acad. Sci. 104:6388-6393 (2007); Fuchs et al,
Proc. Natl. Acad. Sci. Published Online before Print Aug. 23,
2010). Since nucleic acid binding agents (scavengers) described
herein can bind RNA and DNA molecules and shield them from other
potential binding partners, such agents can be employed to inhibit
the ability of DNA and RNA molecules to bind and activate
coagulation factors and platelets. In so doing, these RNA/DNA
scavengers can be utilized to limit nucleic acid-induced
pathological blood coagulation. Thus nucleic acid binding agents
(scavengers) described herein represent novel entities for
preventing the induction and progression of a variety of thrombotic
disorders including myocardial infarction, stroke and deep vein
thrombosis.
[0033] The nucleic acid binding agents of the invention, or
pharmaceutically acceptable salts thereof, can be administered to
the patient via any route such that effective levels are achieved
in, for example, the bloodstream. The optimum dosing regimen will
depend, for example, on the nucleic acid binding agent, the patient
and the effect sought. Typically, the nucleic acid binding agent
will be administered orally, transdermally, IV, IM, IP or SC. The
nucleic acid binding agent can also be administered, for example,
directly to a target site, for example, directly to a tumor (e.g.,
a brain tumor) when cancer is the disease to be treated.
Advantageously, the nucleic acid binding agent is administered as
soon as clinical symptoms appear and administration is repeated as
needed.
[0034] The nucleic acid binding agents (including nucleic acid
binding polymers incorporated into microparticles or nanoparticles
or beads), or pharmaceutically acceptable salts thereof, can be
formulated with a carrier, diluent or excipient to yield a
pharmaceutical composition. The precise nature of the compositions
of the invention will depend, at least in part, on the nature of
the nucleic acid binding agent and the route of administration.
Optimum dosing regimens can be readily established by one skilled
in the art and can vary with the nucleic acid binding agent, the
patient and the effect sought.
[0035] Proteinaceous nucleic acid binding agents of the invention
can also be produced in vivo following administration of a
construct comprising a sequence encoding the proteinaceous nucleic
acid binding agent (Harrison, Blood Rev. 19(2):111-23 (2005)).
[0036] It will be appreciated that the treatment methods of the
present invention are useful in the fields of both human medicine
and veterinary medicine. Thus, the patient (subject) to be treated
can be a mammal preferably a human. For veterinary purposes the
subject can be, for example, a farm animal such as a cow, pig,
horse, goat or sheep, or a companion animal such as a dog or a
cat.
[0037] The invention also relates to methods of identifying nucleic
acid binding agents suitable for use in the above-described
methods. In one embodiment, endosomal TLR-containing cells,
preferably, mammalian cells (e.g., mammalian macrophage cells in
culture), are incubated with a first endosomal TLR agonist (e.g.,
CpG DNA or single or double stranded RNA or nucleic acid-containing
particles) in the presence and absence of a test agent. Following
incubation, a culture supernatant sample can be taken and analyzed
for the presence of a product of an intracellular signaling event
initiated by TLR activation, for example, one or more cytokines
(e.g., TNF.alpha. and/or IL-6). These steps can be repeated with an
endosomal TLR agonist having a sequence, chemistry and/or structure
different from that of the first agonist. A test agent that
inhibits endosomal TLR agonist activation, preferably, in a manner
independent of the sequence, chemistry and/or structure of the
endosomal TLR agonist used, (that inhibition of activation being
evidenced by inhibition of production of a product of an
intracellular signaling event initiated by TLR activation (e.g.,
cytokine production) (e.g., in a dose dependent manner)) can then
be tested in vivo, for example, in mice, to further assess its
suitability for use in the methods described herein.
[0038] Certain aspects of the invention can be described in greater
detail in the non-limiting Example that follows.
EXAMPLE
Experimental Details
[0039] Animal and cell line studies. 8-9 weeks old C57BL/6 mice
were purchased from the Jackson Laboratory (Bar Harbor, Me.). The
murine macrophage cell line, Raw264.7 was obtained from ATCC
(Manassas, Va.).
[0040] Cytokine production of murine macrophage. 1.times.10.sup.6
Raw264.7 cells were cultured in complete medium including DMEM with
10% heat-inactivated. FBS, penicillin, streptomycin and L-glutamine
(2 mM) (all from Invitrogen, Carlsbad, Calif.) in a 24-well culture
plate at 37.degree. C. in a humidified atmosphere with 5% CO.sub.2.
To study TLR activation, the complete medium was supplemented with
phosphorothioate B-type CpG DNA 1668 (5'-TCCATGACGTTCCTGATGCT-3'),
a phosphorothioate GpC DNA 1720 (5'-TCCATGAGCTTCCTGATGCT-3') as a
control CpG DNA (both from IDT, Coralville, Iowa) or a mimetic of
viral dsRNA, poly I:C (Amersham/GE Healthcare, Piscataway, N.J.) at
various concentrations. Bacterial LPS serotype 026:B6 (100 ng/ml)
(Sigma-Aldrich, Saint Louis, Mo.) activating TLR4 were used as a
non-nucleotide-based TLR ligand. To block TLR activation CDP
(Calando Pharmaceuticals, Pasadena, Calif.), protamine (APP,
Schaumburg, Ill.), PPA-DPA, PAMAM, poly-L-lysine or HDMBr
(polybrene) (kindly provided by Dr. Kam W. Leong, Duke University,
Durham, N.C.) at various concentrations were simultaneously treated
with either CpG DNA or poly IC. After 18 hours of incubation,
culture supernatant were collected and stored at -80.degree. C. for
later analyses. The production of TNF.alpha. and IL-6 were analyzed
with BD OptEIA.TM. ELISA sets (BD Biosciences, Franklin Lakes,
N.J.) by following the manufacturer's instructions.
[0041] Co-stimulatory molecule expression on macrophage.
1.times.10.sup.6 Raw264.7 cells were cultured with phosphate buffer
saline (PBS), GpC DNA (2 .mu.M) or CpG DNA (2 .mu.M). To block
binding of CpG DNA and TLR9 CDP, HDMBr, PAMAM, PPA-DPA,
poly-L-lysine or protamine (20 .mu.g/ml each) were co-treated with
CpG DNA. After 18 hours, cells were detached from plates by
treatment of 0.05% Trypsin-EDTA (Invitrogen), washed twice with PBS
and stained with either phycoerythrin (PE)-anti-mouse CD86 (GL1) or
PE-rat IgG2a, .kappa. as an isotype control (all from eBioscience,
San Diego, Calif.). Cells were washed with PBS, fixed with 4%
formaldehyde, and analyzed on a FACS Caliber (BD Biosciences).
[0042] Mouse TLR-mediated acute liver injury. TLR3 or TLR9-mediated
acute liver injury in a D-galactosamine-sensitized mice was
performed as previously described (Alexopoulou et al Nature
413:732-738 (2001), Duramad et al, J. Immunol. 174:5193-5200
(2005)). Briefly, C57BL/6 mice were injected intraperitoneally
(i.p.) with PBS (100 .mu.l), CpG DNA (25 to 51 .mu.g), GpC DNA (50
.mu.g) or poly I:C (50 to 200 82 g) with or without
D(+)Galactosamine, Hydrochloride (D-GalN) (EMD Biosciences, La
Jolla, Calif.) (20 mg). Five to ten minutes after toxin challenge,
cationic molecules (200 to 800 .mu.g) were injected i.p. Viability
of mice was monitored for a week.
[0043] Histopatholoy. Liver lobes were excised from mice 24 hours
after injection of CpG+D-GalN with or without cationic molecules.
The liver specimens were fixed with 4% formaldehyde, embedded in
OCT and sectioned at a thickness of 20 .mu.m before staining with
hematoxylin and eosin for light microscopic examination.
[0044] Statistical Analysis. The difference of cytokine production
among experimental groups was compared by the paired two-tailed
Student's t test analyzed with Microsoft Office Excel 2003.
Significance of survival was determined by the log-rank test
analyzed with GraphPad Prism.RTM. Version 4.0b. A probability of
less than 0.05 (P<0.05) was used for statistical
significance.
Results
[0045] Six agents known to bind nucleic acids were evaluated for
their ability to attenuate endosomal TLR responses:
.beta.-cyclodextrin-containing polycation (CDP),
polyphosphoramidate polymer (PPA-DPA), polyamidoamine dendrimer,
1,4-diaminobutane core, G3 (PAMAM), poly-L-lysine, hexadimethrine
bromide (HDMBr; also known as polybrene) and protamine. Five of the
compounds inhibited polyinosinic-polycytidylic acids (poly I:Cs), a
dsRNA activator of TLR3, stimulation of macrophages as measured by
TNF.alpha. and IL-6 production and three prevented inflammatory
cytokine production from the cells stimulated with unmethylated CpG
DNA, a TLR9 agonist (FIG. 1A). The CpG DNA-inhibitory cationic
polymers also impeded the up-regulation of co-stimulatory molecules
expressed on macrophages (FIG. 1B). Interestingly, the inhibitors
could be administered up to 4 hours after the CpG DNA and still
significantly reduce TNF.alpha. and IL-6 production from
macrophages (FIG. 2). Pre-treatment of macrophages with CDP,
however, did not alter the ability of the cells to produce
inflammatory cytokines (FIG. 2). By contrast, the nucleic
acid-binding cationic polymers did not inhibit LPS-mediated
activation of macrophages, which indicates that they specifically
interfere with recognition of nucleic acids by TLRs.
[0046] The nucleic acid-binding polymers inhibit TLR3 and TLR9
activation in a dose-dependent manner. A dose-escalation study
demonstrated that 8 to 12 .mu.g/ml of the polymers, CDP, HDMBr and
PAMAM, which inhibited the activation of both TLR3 and TLR9, can
inhibit inflammatory cytokine production by greater than 95% from
macrophages treated with CpG DNAs (1 .mu.M) and 5 to 40 .mu.g/ml of
these same polymers can reduce cytokine production by greater than
95% from cells treated with poly I:C (10 .mu.g/ml) (FIGS. 3A and
5).
[0047] One concern about using cationic polymers as therapeutic
agents is their potential toxicity since certain cationic carriers
are know to have high cytotoxicity (Hunter, Adv. Drug Deliv. Rev.
58:1523-1531 (2006)). Poly L-lysine (10-40 .mu.g/ml) has been shown
to induce significant apoptosis of mammalian cells (Symonds et al,
FEBS Lett. 579:6191-6198 (2005)). By contrast, the LD.sub.50 of CDP
is 200 mg/kg in mice (Hwang et al, Bioconjug. Chem. 12:280-290
(2001)). Therefore, the cytotoxicity of the cationic polymers used
in the current study was evaluated on macrophages (FIG. 6). Poly
L-lysine and PPA-DPA induced over 50% cell death at approximately
20 and 40 .mu.g/ml, respectively, while PAMAM, protamine and HDMBr
induced over 50% cell death at about 160, 280 and 600 .mu.g/ml,
respectively. The CDP polymer was well tolerated on macrophages. In
mice injected with the CDP, HDMBr and PAMAM at 40 mg/kg, no adverse
effects on viability were observed (data not shown). In summary,
poly L-lysine and PPA-DPA have a relatively high cytotoxicity while
PAMAM, HDMBr and CDP have much less toxicity in vitro and in
vivo.
[0048] Finally, the ability of the nucleic acid-binding polymers to
limit endosomal TLR activation in vivo was evaluated. It has been
shown that injection of CpG DNA or poly I:C into mice sensitized
with D-galactosamine (D-GalN) induces a TLR-mediated acute
inflammatory response which can result in liver damage and death
(Alexopoulou et al, Nature 413:732-738 (2001), Duramad et al, J.
Immunol. 174:5193-5200 (2005)). Consistent with previous reports,
greater than 90% of the mice died by 48 hours following
administration of D-GalN and CpG DNA or poly I:C while none of the
mice injected with D-GalN alone, CpG DNA alone or D-GalN and
control GpC DNA died. Strikingly, administration of one of three
different nucleic acid-binding polymers, CDP, HDMBr or PAMAM,
immediately following D-GalN and CpG DNA or poly I:C resulted in
significant protection of the animals in a dose-dependent manner
and reduced mortality by almost 100% in several cases (FIGS. 4A and
4B). Histological examination of livers from treated mice also
demonstrated that inflammation and associated hemorrhage were
greatly reduced in the polymer treated animals (FIG. 4C).
[0049] Cationic polymers are commonly used for gene or siRNA
delivery and are designed to facilitate cellular internalization
and endosomal escape (Morille et al, Biomaterials.29:3477-3496
(2008)). Because they traffic through the endosomal compartment,
cationic lipids have been used to deliver siRNAs and immune
stimulatory ssRNAs to activate endosomal TLR7 or TLR8 (Judge et al,
Nat. Biotechnol. 23:457-462 (2005), Sioud, J. Mol. Biol.
348:1079-1090 (2005)). Moreover, synthetic ssRNAs or mRNAs
pre-condensed with protamine induced inflammatory cytokine
production in human PBMCs via activation of TLR7 or TLR8 (Scheel et
al, Eur. J. Inununol. 35:1557-1566 (2005)). Similarly, it was
observed that treatment with protamine did not block but
significantly enhanced inflammatory cytokine production from cells
stimulated with poly I:C (FIG. 1A). In striking contrast, it was
observed in the above-described studies that the cationic polymers,
CDP, HDMBr and PAMAM, neutralize the ability of nucleic acid-based
TLR3 and TLR9 ligands to activate their cognate TLRs and induce
inflammatory responses. Several potential explanations exist for
these observed differences. In the present studies, cells were
treated with endosomal TLR ligands and cationic polymers separately
while in the previous studies immune stimulatory RNAs were
pre-condensed with cationic molecules before exposure to cells.
Thus, the pre-condensation of RNA and cationic molecules could
generate a particle that might be efficiently endocytosed. By
contrast, nucleic acids, that are not assembled into particles, may
be only poorly taken up by cells and addition of the polymers would
form small complexes not recognized by the cell. To test this
possibility, the cellular uptake of CpG DNAs was evaluated.
Unexpectedly, treatment with CDP enhanced cellular uptake of CpG
DNAs, even though this did not lead to endosomal TLR9 activation
(FIG. 7). The reason why CpG delivered into cells in this manner
does not elicit a TLR response is unclear. The polymer may alter
endosomal maturation and thus TLR signaling or the polymer may
directs the CpG into a distinct intracellular trafficking pathway
(Morille et al, biomaterials 29:3477-3496 (2008), Krieg, Annu. Rev.
Immunol. 20:709-760 (2002), Jozefowski et al, J. Leukoc. Biol.
80:870-879 (2006)). Further investigation will be required to
understand how cationic polymers neutralize nucleic acid activation
of endosomal TLRs and why some cationic polymers are more effective
than others at impeding such responses.
[0050] In summary, nucleic acid-binding polymers can simultaneously
limit the activation of multiple endosomal TLRs. As such, these
polymers represent promising therapeutic agents for treating
patients with inflammatory diseases and autoimmune diseases.
Additional preclinical and clinical studies will evaluate this
possibility.
[0051] All documents and other information sources cited above are
hereby incorporated in their entirety by reference.
* * * * *